Bottom Line:
The difficulty, however, arises from the limited complementarity of metazoan miRNAs to their targets, with the interaction requiring as few as six nucleotides of the miRNA seed sequence.To identify miR-294 targets, we used Dicer1- ES cells, which lack all endogenous mature miRNAs, and introduced just miR-294 into these ES cells.Our data suggest that miR-294 may promote pluripotency by regulating a subset of c-Myc target genes and upregulating pluripotency-associated genes such as Lin28.

ABSTRACTMouse Embryonic Stem (ES) cells express a unique set of microRNAs (miRNAs), the miR-290-295 cluster. To elucidate the role of these miRNAs and how they integrate into the ES cell regulatory network requires identification of their direct regulatory targets. The difficulty, however, arises from the limited complementarity of metazoan miRNAs to their targets, with the interaction requiring as few as six nucleotides of the miRNA seed sequence. To identify miR-294 targets, we used Dicer1- ES cells, which lack all endogenous mature miRNAs, and introduced just miR-294 into these ES cells. We then employed two approaches to discover miR-294 targets in mouse ES cells: transcriptome profiling using microarrays and a biochemical approach to isolate mRNA targets associated with the Argonaute2 (Ago2) protein of the RISC (RNA Induced Silencing Complex) effector, followed by RNA-sequencing. In the absence of Dicer1, the RISC complexes are largely devoid of mature miRNAs and should therefore contain only transfected miR-294 and its base-paired targets. Our data suggest that miR-294 may promote pluripotency by regulating a subset of c-Myc target genes and upregulating pluripotency-associated genes such as Lin28.

pgen-1001163-g002: Global gene expression changes in the INPUT.(A) RNA-Sequencing (RNA-Seq) of the INPUT (Total RNA) follows the microarray trend in gene expression. The y-axis represents the RNA-Seq ratio of the INPUT from Dicer1Δ/Δ+Ago2-myc ES cells transfected with miR-294 vs. cel-239b. Higher values mean enriched in cel-239b vs. miR-294. The x-axis represents the fold change in gene expression as measured by microarray. A positive value indicates upregulation of genes in miR-294-transfected cells and a negative value indicates downregulation of genes in miR-294-transfected cells. (B) Overrepresentation of 6-mer (position 2-7) seed matches to miR-294 in the INPUT of Dicer1Δ/Δ+Ago2-myc ES cells transfected with miR-294. The x-axis represents the sorted gene list from most downregulated (left) to most upregulated (right). The y-axes show the significance for each word.

Mentions:
A comparison of the global gene expression changes detected by microarray and by RNA-Sequencing of the INPUT revealed a similar trend (Figure 2A). Furthermore, there was overrepresentation of the miR-294 seed sequence in the most downregulated genes of the sequenced INPUT (Figure 2B), indicating that miR-294 was functional and that the transfection efficiency was sufficiently high (Figure S3B) to bring about gene expression changes. An examination of the IP vs. INPUT ratios for each sample revealed an overall greater dynamic range of enrichment for samples transfected with miR-294 compared to cel-239b (Figure S4). The pattern was similar for the catalytically-inactive Ago2-myc. This result was expected since miRNA-directed slicing of the target mRNA is thought to occur rarely in metazoans [12]. However, the overall extra enrichment could not be accounted for by the overrepresentation of miR-294 seed sequence matches in the 3′ UTRs of enriched genes (Figure 3A). There is a surprising general tendency for hexamers with a higher GC content to be overrepresented in the 3′ UTRs of enriched transcripts (Figure 3A and 3B). This effect is also observed in samples transfected with the control cel-239b (Figure 3C). However, the GC effect is markedly stronger in the samples transfected with miR-294.

pgen-1001163-g002: Global gene expression changes in the INPUT.(A) RNA-Sequencing (RNA-Seq) of the INPUT (Total RNA) follows the microarray trend in gene expression. The y-axis represents the RNA-Seq ratio of the INPUT from Dicer1Δ/Δ+Ago2-myc ES cells transfected with miR-294 vs. cel-239b. Higher values mean enriched in cel-239b vs. miR-294. The x-axis represents the fold change in gene expression as measured by microarray. A positive value indicates upregulation of genes in miR-294-transfected cells and a negative value indicates downregulation of genes in miR-294-transfected cells. (B) Overrepresentation of 6-mer (position 2-7) seed matches to miR-294 in the INPUT of Dicer1Δ/Δ+Ago2-myc ES cells transfected with miR-294. The x-axis represents the sorted gene list from most downregulated (left) to most upregulated (right). The y-axes show the significance for each word.

Mentions:
A comparison of the global gene expression changes detected by microarray and by RNA-Sequencing of the INPUT revealed a similar trend (Figure 2A). Furthermore, there was overrepresentation of the miR-294 seed sequence in the most downregulated genes of the sequenced INPUT (Figure 2B), indicating that miR-294 was functional and that the transfection efficiency was sufficiently high (Figure S3B) to bring about gene expression changes. An examination of the IP vs. INPUT ratios for each sample revealed an overall greater dynamic range of enrichment for samples transfected with miR-294 compared to cel-239b (Figure S4). The pattern was similar for the catalytically-inactive Ago2-myc. This result was expected since miRNA-directed slicing of the target mRNA is thought to occur rarely in metazoans [12]. However, the overall extra enrichment could not be accounted for by the overrepresentation of miR-294 seed sequence matches in the 3′ UTRs of enriched genes (Figure 3A). There is a surprising general tendency for hexamers with a higher GC content to be overrepresented in the 3′ UTRs of enriched transcripts (Figure 3A and 3B). This effect is also observed in samples transfected with the control cel-239b (Figure 3C). However, the GC effect is markedly stronger in the samples transfected with miR-294.

Bottom Line:
The difficulty, however, arises from the limited complementarity of metazoan miRNAs to their targets, with the interaction requiring as few as six nucleotides of the miRNA seed sequence.To identify miR-294 targets, we used Dicer1- ES cells, which lack all endogenous mature miRNAs, and introduced just miR-294 into these ES cells.Our data suggest that miR-294 may promote pluripotency by regulating a subset of c-Myc target genes and upregulating pluripotency-associated genes such as Lin28.

ABSTRACTMouse Embryonic Stem (ES) cells express a unique set of microRNAs (miRNAs), the miR-290-295 cluster. To elucidate the role of these miRNAs and how they integrate into the ES cell regulatory network requires identification of their direct regulatory targets. The difficulty, however, arises from the limited complementarity of metazoan miRNAs to their targets, with the interaction requiring as few as six nucleotides of the miRNA seed sequence. To identify miR-294 targets, we used Dicer1- ES cells, which lack all endogenous mature miRNAs, and introduced just miR-294 into these ES cells. We then employed two approaches to discover miR-294 targets in mouse ES cells: transcriptome profiling using microarrays and a biochemical approach to isolate mRNA targets associated with the Argonaute2 (Ago2) protein of the RISC (RNA Induced Silencing Complex) effector, followed by RNA-sequencing. In the absence of Dicer1, the RISC complexes are largely devoid of mature miRNAs and should therefore contain only transfected miR-294 and its base-paired targets. Our data suggest that miR-294 may promote pluripotency by regulating a subset of c-Myc target genes and upregulating pluripotency-associated genes such as Lin28.